Communication
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Slight pH Fluctuations in the Gold Nanoparticle
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Synthesis Process Influence the Performance of the
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Citrate Reduction Method
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Braulio Contreras-Trigo1, Víctor Díaz-García1, Enrique Guzmán-Gutierrez2, Ignacio Sanhueza3,
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Pablo Coelho3, Sebastián Godoy3, Sergio Torres3 and Patricio Oyarzún1,*
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1 Facultad de Ingeniería y Tecnología, Universidad San Sebastián, Lientur 1457, Concepción 4080871, Chile;
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[email protected] (B.C.), [email protected] (V.D).
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2 Facultad de Ciencias de la Salud, Universidad San Sebastián, Lientur 1457, Concepción 4080871, Chile;
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[email protected] (E.G.),
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3 Departamento de Ingeniería Eléctrica, Facultad de Ingeniería, Universidad de Concepción;
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[email protected] (I.S.), [email protected] (P.C.), [email protected] (S.G.), [email protected]
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(S.T.)
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* Correspondence: [email protected]; Tel.: +56-41-2487962
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Abstract: Gold nanoparticles (AuNPs) are currently under intense investigation for biomedical and
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biotechnology applications, thanks to their ease in preparation, stability, biocompatibility, multiple
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surface functionalities and size-dependent optical properties. The most commonly used method for
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AuNPs synthesis in aqueous solution is the reduction of tetrachloroauric acid (HAuCl4) with
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trisodium citrate. We observed variations in the pH and concentration of the gold colloidal
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suspension synthesized under standard conditions, verifying a reduction in the reaction yield by
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around 46% from pH 5.3 (2.4 nM) to pH 4.7 (1.29 nM). Citrate-capped AuNPs were characterized
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by UV-visible spectroscopy, TEM, EDS and zeta-potential measurements, revealing a linear
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correlation between pH and the concentration of the generated AuNPs. This result can be attributed
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to the adverse effect of protons both on citrate oxidation and on citrate adsorption onto the gold
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surface, which is required to form the stabilization layer. Overall, this study provides insight into
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the effect of the pH over the synthesis performance of the method, which would be of particular
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interest from the point of view of large-scale manufacturing processes.
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Keywords: Gold nanoparticles; Citrate reduction method; pH-effect; concentration
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1. Introduction
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Gold nanoparticles (AuNPs) have emerged as a promising platform for a growing number of
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biomedical and biotechnology applications in the fields of sensing [1], molecular diagnostic [2],
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therapeutic [3] and imaging [4], owing to their stability, biocompatibility, remarkable
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physicochemical properties and easy surface functionalization with a wide range of ligands [5, 6]. A
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prominent optic feature of AuNPs arises from the collective oscillation of the conduction electrons in
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the presence of an incident light, the so-called surface plasmon resonance (SPR) [7]. This phenomenon
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causes a sharp and intense absorption band in the visible range, which can be readily tuned by
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varying the particle size, shape and the surrounding physicochemical environment [8]. Thus,
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free colorimetric sensors based on AuNPs (nanobiosensors) have been widely proposed as a
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promising analytical for selective binding and detection of chemical and biological targets, including
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metal ions [9], antibiotics [10], mycotoxins [11], as well as a large number of microorganisms [12].
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As concerns the preparation of the colloidal gold nanoparticles, various chemical routes, including
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the use of chemical reductants [13] and several photochemical methods based on UV irradiation [14],
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γ-irradiation [15] and laser irradiation [16], have been widely studied for different purposes of
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application. However, the classical “citrate reduction method” proposed by Turkevich in 1951 [17],
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and latter modified by Frens in 1973 [18], remains the most widely employed synthesis procedure,
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since AuNPs can be produced in a straightforward manner to obtain highly stable monodisperse
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particles with uniform spherical shape and narrow-size distributions ranging between 10 - 20 nm in
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diameter. This method is based on the aqueous-phase reduction of an Au3+ precursor (HAuCl4; gold
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salt) with sodium citrate near the boiling point of the reaction mixture, which produces a stable
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solution of metallic gold nanoparticles (colloidal gold) [19].
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Citrate ions play a key role, since they act both as reductant, converting gold ions (Au3+) into gold
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atoms (Au0), and as a protective agent that stabilizes the formed nanoparticles, preventing particle
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growth and aggregation via electrostatic repulsion (citrate capped AuNPs) [20]. Thus, at high citrate
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concentrations, smaller particles are covered and stabilized by this ion, while at low concentrations
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particle growth continues due to incomplete coverage, leading to the formation of AuNPs with larger
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particle size. A third role of citrate is as a mediator of the reaction mixture pH, through which it has
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a dramatic effect on the size, polydispersity and morphology of the resulting AuNPs [21, 22]. pH
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control on the citrate reduction method has been widely explored regarding its relationship with the
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size distribution of the AuNPs [23]. However, up to now the correlation of this parameter with the
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concentration of the resulting nanoparticles had not been described, which is of particular interest for
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citrate mediated synthesis of colloidal AuNPs to meet large scale manufacturing criteria [24].
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The acid-base behavior of the AuNPs is provided by the citrate layer, which imparts a negative charge
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onto the colloidal particle surface. Citrate is a tricarboxylic acid (polyanion) with three pKa values
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(pKa1=3.06, pKa2=4.74 and pKa3=5.4) [25], which participate in the following chemical equilibria:
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In this study we investigated the relationship between typically occurring pH fluctuations of the
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colloidal suspension (between 4.5 and 5.3) and the concentration of the gold nanoparticles, providing
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valuable information regarding the synthesis performance of the method. AuNPs were characterized
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in terms of their concentration (optical density determinations), morphology and surface charge, by
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carrying out transmission electron microscopy (TEM) examination, scanning electron microscopy
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with energy dispersive X-ray spectroscopy (EDS) and zeta potential analysis.
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2. Materials and Methods
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2.1. Preparation of gold nanoparticles (AuNPs)
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Synthesis of AuNPs was carried out according to the procedures described according to the
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citrate reduction method [17, 18]. Briefly, 0.0394 g of tetrachloroauric acid (HAuCl4⋅3H2O) (Merk,
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USA) was dissolved in 100 mL of nanopure water (18 MΩ of resistance) in a two-neck round flask (1
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mM HAuCl4). The resulting solution was heated to boiling under stirring and refluxed. Then, 10 mL
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of a 38.8 mM trisodium citrate solution was preheated and quickly added to the boiling solution of
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HAuCl4 under vigorous stirring. After the solution turned from pale yellow to black and to deep red,
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it was refluxed for additional 30 min and subsequently cooled to room temperature without stirring
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μm) and preserved in the dark at 4 °C for subsequent pH determination and characterization of the
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AuNPs.
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2.2. AuNP concentration
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The concentration of gold synthesized under different pH conditions was determined by
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Vis spectroscopy using an EpochTM Microplate Spectrophotometer (Bio-Tek Instruments, Winooski,
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VT, USA). 100 μL of each sample were transferred into the microplate wells and the absorption
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spectra was recorded in the visible region (400 to 700 nm). The absorption maximum at the SPR band
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(520 nm) was employed to calculate the AuNP concentration according to the Beer-Lambert law, by
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using an extinction coefficient (ε) of 2.01x108 M-1 cm-1 [26].
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2.3. Electronic microscopy analyses
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The morphology and size of the AuNPs was determined by transmission electron microscopy
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with 4 Å resolution (TEM; JEOL-JEM 1200EX-II, Tokyo, Japan). Carbon-coated 100 mesh copper grids
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were dipped into a small drop of each AuNP solution, which were subsequently retracted and
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allowed to dry in air at room temperature. Particle sizes and frequency histograms were obtained by
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measuring the diameter of 100 nanoparticles using the ImageJ software [27], while the percentage of
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spherical particles was determined through visual inspection of 100 nanoparticles. In addition,
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elemental analysis of the gold colloidal suspensions was carried out by energy-dispersive X-ray
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analysis (EDS) using scanning electron microscopy (SEM; Etec Autoscan U-1).
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2.4. Surface charge characterization (pZ)
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Zeta-potential measurements of AuNPs were measured on a zeta-potentiometer (Nano-ZS90,
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Malvern Instruments, Westboroug) at room temperature and scattering angle of 90°. A diluted
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suspension of AuNPs (100 µL diluted to 1 mL nanopure water) were employed for the analyses. The
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Malvern Zetasizer Software version 7.12 was employed to analyze the collected data.
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3. Results and Discussion
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3.1. pH effect on the concentration
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Figure 1 presents the absorption spectra of the AuNPs samples synthesized at three pH values (4.7,
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5.0 and 5.3), showing the typical curve with a characteristic maximum at 520 nm associated to the
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SPR band. However, differences in the absorbance values at this peak revealed that pH of the
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synthesis medium had a relevant effect on the resulting concentration of the nanoparticles. The
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AuNPs concentration varied in a directly proportional manner as a function of the pH (positive
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correlation), reaching at pH 5.3 about 1.8 fold the concentration (2.4 nM) than that obtained at pH 4.7
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(1.29 nM) (see Table 1). This correlation is highly linear in the pH range monitored (4.7 - 5.3), with a
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correlation coefficient (r2) of 0.9987.
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Figure 1. Absorption spectra of AuNPs obtained by the citrate reduction method at pHs 4.7 (red), 5.0
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(blue) and 5.3 (green). At the top right corner a linear correlation curve between pH and AuNP
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concentration is presented. O.Ds correspond to mean values calculated from independent
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determinations (n=2).
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3.2. AuNPs characterization
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TEM analysis of the AuNPs samples proved the nanoparticles have comparable diameters between
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13.9–15.5 nm for the tree monitored pH values, with similar morphology distribution (Figure 2; Table
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1). The percentage of spherical nanoparticles was slightly higher at pH 5.3 (77%), in comparison with
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that at pH 5.0 (57%) and pH 4.7 (62%), which would favor shape homogeneity of the nanoparticles.
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By contrast, commercial AuNPs showed the least sphericity (51%). These observations are consistent
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with the similar optical behavior exhibited by the nanoparticle suspensions, which share identical
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wavelength associated to the SPR peak (UV-Vis spectrograms, Figure 1).
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Figure 2. Transmission electron microphotographs of AuNPs at pH 4.7 (a), 5.0 (b) and 5.3 (c), and
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commercial AuNPs (pH 5.0) with an average diameter of 16.42 ± 1.76 nm (d). Histograms with the
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respective particle size distributions are included within each microphotography, showing on top
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right corner the percentage of spherical nanoparticles.
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In addition, energy dispersive X-ray spectrometry (EDS) elemental analysis confirmed the presence
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of Au, C, O and Na forming part of the citrate layer adsorbed on the AuNPs (Figure 3). Sodium ions
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play an important role in stabilizing high coverages of the gold surface by carboxylate anions [28].
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Figure 3. EDS spectra of gold colloidal suspensions at pH 4.7 (a), 5.0 (b) and 5.3 (c). Higher peaks
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correspond to the silicon from the glass supports.
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3.3 Surface charge analysis
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Under standard experimental conditions employed in the present study the binding of citrate ions
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onto the surface of AuNPs occurs electrostatically through negative oxygen atoms of carboxylic
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groups (oxyanions) at the ends of the citrate molecule [28]. Thus, at pH 4.7 (close to pKa2) similar
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amounts of H2Cit-1 and HCit-2 implies a lesser availability of deprotonated carboxylic groups, while
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at pH 5.3 (close to pKa3) the dominant species HCit-2 and Cit-3 would determine a greater capability
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of citrate anions to coordinate to the metal surface.
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The negative charge of citrate-stabilized AuNPs was confirmed through zeta potential analysis (pZ),
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revealing the influence of pH on the surface charge from the distribution of the pZ values (Figure 4).
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Thus, as the monitored pH fluctuated from 4.7 to 5.3, the shape of the curves reveals a progressive
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narrowing on the dispersion of the pZ values of the AuNPs, which accounts for a greater stabilization
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of the nanoparticles as a consequence of the presence of mostly deprotonated citrate anions.
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Table 1. Zeta potential, concentration and diameter of AuNPs at pHs 4.7, 5.0 and 5.3.
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Figure 4. Zeta potential distribution (mV) of citrate-capped AuNPs synthesized at pHs 4.7 (red), 5.0
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(blue) and 5.3 (green).
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The lowest dispersion values were determined at pHs 5.0 and 5.3 (-44.9 ± 5.1 mV and -45.7 ± 7.6 mV,
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respectively), with curves showing single and narrow peaks. Similarly, a recent study showed that a
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pH value of 5 was optimal to produce gold nanoparticles that are highly monodisperse and spherical
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in shape, with a detrimental effect on the size polydispersity at lower pH values [29]. By contrast, the
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highest pZ dispersion was observed at pH 4.7 (-42.2 ± 35.1 mV), showing a flat distribution curve
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with three minor peaks that accounts for different populations of citrate-capped AuNPs having a
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wide range of surface charges. Importantly, the right tail of this curve mostly falls into the region of
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pH Zeta Potential mean (mV)
Peak 1 (mV)
Peak 2 (mV)
Peak 3 (mV)
Concentration (nM)
Diameter (nm)
4.7 -42.2 ± 35.1 -58.5 ± 15.2 -26.7 ± 8.2 16.8 ± 7.1 1.29 ± 0.09 13.92 ± 1.45
positive surface charge values (minor peak at 16.87± 7.1 mV), revealing a loss on the citrate coverage
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of the gold surface. Therefore, these results provide support to an expected lesser tendency of
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partially protonated citrate ionic species to coordinate with the AuNPs at pH 4.7.
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In order to understand the effect of pH on the reaction, the role of citrate (and protons) must be
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considered both in terms of the redox reaction forming AuNPs (citrate as oxidizer), as well as from
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the point of view of its protectant role (citrate as stabilizer). First, looking at the standard reaction
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mechanism, the initial step of this multiple-step process is the oxidation of citrate yielding dicarboxy
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acetone (Eq. 1). The second step consist of reduction of auric salt (Au3+) to aurous salt the (Au1+) by
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accepting the electrons from the citrate oxidation reaction (Eq. 2), and the final step is the
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disproportionation of aurous species to gold atoms (Au0) (Eq. 3).
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OCOCH C OH COO → OCOCH C = O + CO + H + 2e Eq. 1
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AuCl + 2e → AuCl + 2Cl Eq. 2
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3AuCl → 2Au + AuCl Eq. 3
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The formation of an intermediate pentacoordinate complex of Au3+ species with
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dicarboxyacetone,has been proposed, which subsequently decarboxylates to give Au+ species [28].
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However, the overall stoichiometry of the reduction reaction can be represented as:
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2AuCl + 3 OCOCH C OH COO → 2Au + 3 OCOCH C = O + 6Cl + 3H + 3CO Eq. 4
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In accordance with Le Chatelier’s principle, as the concentration of protons increases in the solution,
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the tendency of citrate to oxidize decreases, as well as the availability of electron to reduce the
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gold(III) chloride and to form AuNPs. In the second place, coordination bondings between the gold
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surface and adsorbed molecules of citrate consist of carboxyl oxygens that contribute the electron
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pairs forming covalent bonds [28]. The complexity of the structural arrangement of the citrate layers
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on AuNPs has been analyzed in several recent studies, showing that citrate could coordinate to the
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gold through several different binding modes (geometries) that are mostly dictated by one or two of
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the terminus carboxyl groups [30-32]. However, to promote the intermolecular interactions it is
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required for citrate to diffuse on the gold surface in a preferential fully deprotonated form, since
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protonated carboxylate groups does not readily adsorb on gold due to electrochemical impediments
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[33]. Accordingly, the protonation of citrate terminus sites is considered to discourage citrate binding
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to the gold surface [31], in agreement with our observation of the detrimental effect of pH fluctuations
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on coverage, stabilization and production of the AuNPs.
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4. Conclusions
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We demonstrated that under standard experimental conditions of the citrate reduction method, gold
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nanoparticles are produced in variable concentrations, showing a pH-dependent linear correlation in
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the range monitored between pH 4.7 and 5.3 (typical pH fluctuation). The AuNP synthesis
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performance reduced by 46% from 2.4 nM (pH 5.3) to 1.29 nM (pH 4.7), without alteration of size and
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morphology. The physical characterization of the nanoparticles suggests that slight fluctuations of
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pH during synthesis can have an adverse effect on citrate oxidation, as well as on the availability of
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negative charges from carboxylate groups required for an optimal coverage, stabilization and
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production of the nanoparticles. Overall, these results provide insight into the effect of the pH over
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the AuNP synthesis performance of the method, which would be of particular interest for further
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studies optimizing the reaction conditions in large-scale manufacturing processes.
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Acknowledgments: This study was supported by The Scientific and Technological Development Support Fund
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of Chile (FONDEF ID16I10221) and Dirección General de Investigación Universidad San Sebastián, Chile.
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Author Contributions: B.C., V.D., E.G. and P.O. conceived and designed the experiments; B.C. and V.D.
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performed the experiments; B.C., V.D., E.G., I.S., P.C., S.G., S.T. and P.O. analyzed the data; B.C., V.D., E.G. and
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P.O. wrote the paper.
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Conflicts of Interest: The authors declare no conflict of interest.
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